The general process for the manufacture of biomolecules, such as proteins, particularly recombinant proteins typically involves two main steps: (1) the expression of the protein in a host cell, followed by (2) the purification of the protein. The first step involves growing the desired host cell in a bioreactor to effect the expression of the protein. Some examples of cell lines used for this purpose include Chinese hamster ovary (CHO) cells, myeloma (NSO) bacterial cells such as e-coli and insect cells. Once the protein is expressed at the desired levels, the protein is removed from the host cell and harvested. Suspended particulates, such as cells, cell fragments, lipids and other insoluble matter are typically removed from the protein-containing fluid by filtration or centrifugation, resulting in a clarified fluid containing the protein of interest in solution as well as other soluble impurities.
The second step involves the purification of the harvested protein to remove impurities which are inherent to the process. Examples of impurities include host cell proteins (HCP, proteins other than the desired or targeted protein), nucleic acids, endotoxins, viruses, protein variants and protein aggregates.
This purification typically involves several chromatography steps, which can include affinity, ion exchange hydrophobic interaction, etc on solid matrices such as porous agarose, polymeric or glass or by membrane based adsorbers.
One example of a chromatography process train for the purification of proteins involves use of protein-A affinity for the purification of monoclonal antibodies, followed by cation exchange, followed by anion exchange. The protein-A column captures the protein of interest or target protein by an affinity mechanism while the bulk of the impurities pass through the column to be discarded. The protein then is recovered by elution from the column. Since most of the proteins of interest have isoelectric points (pI) in the basic range (8-9) and therefore being positively charged under normal processing conditions (pH below the pI of the protein), they are bound to the cation exchange resin in the second column. Other positively charged impurities are also bound to this resin. The protein of interest is then recovered by elution from this column under conditions (pH, salt concentration) in which the protein elutes while the impurities remain bound to the resin. The anion exchange column is typically operated in a flow through mode, such that any negatively charged impurities are bound to the resin while the positively charged protein of interest is recovered in the flow through stream. This process results in a highly purified and concentrated protein solution.
Other alternative methods for purifying proteins have been investigated in recent years, one such method involves a flocculation technique. In this technique, a soluble polyelectrolyte is added to an unclarified cell culture broth to capture the suspended particulates and a portion of the soluble impurities thereby forming a flocculent, which is subsequently removed from the protein solution by filtration or centrifugation.
Alternatively, a soluble polyelectrolyte is added to clarified cell culture broth to capture the biomolecules of interest, thereby forming a flocculent, which is allowed to settle and can be subsequently isolated from the rest of the solution. The flocculent is typically washed to remove loosely adhering impurities. Afterwards, an increase in the solution's ionic strength brings about the dissociation of the target protein from the polyelectrolyte, subsequently resulting in the resolubilization of the polyelectrolyte into the protein-containing solution.
The main drawback of this flocculation technique is that it requires that the polyelectrolyte be added in the exact amount needed to remove the impurities or capture the biomolecule of interest. If too little flocculent is added, impurities and/or a portion of the target biomolecule (protein, peptide, polypeptide, antibody fragment, etc) will remain in the solution. On the other hand, if too much flocculent is added, the excess polyelectrolyte needs to be removed from the resulting solution. The exact level of impurities in the broth is extremely difficult to predict due to the relatively large degree of variability in the process (from batch to batch) as well as the vast differences between processes to produce different proteins. Removing any excess polyelectrolyte is practically impossible because it is a soluble material and thus it is carried through the process as an undesirable impurity.
In co-pending application U.S. Ser. No. 12/004,314 filed Dec. 20, 2007, a polymer, soluble under certain conditions, such as temperature, pH, salt, light or combinations thereof, is used to bind impurities while in its soluble state and is then precipitated out upon a change in condition (pH or temperature, etc) removing the impurities with it. The biomolecule of interest is then further treated using traditional chromatography or membrane adsorbers and the like.
In co-pending application U.S. Ser. No. 12/004,319 filed Dec. 20, 2007 it was suggested that one would use the clarification process and chemistries of the application mentioned above to provide one with a clarified feedstock and then use the different chemistries and processes of this case to purify the biomolecule of interest.
All of the protein purification technologies discussed above share a common theme, and said theme is to first remove suspended particulates in a first distinct step and then in a second step separate the biomolecules of interest from soluble impurities which are inherent to the process.
In situ product recovery with derivatized magnetic particles is one example of a protein purification technique where the biomolecules of interest can be purified directly from an un-clarified cell culture broth. In this technique, a polymer shell encapsulating a magnetic bead is functionalized with an affinity ligand that seeks out and binds the target protein. A magnetic field is then applied to collect the bead-protein complexes, leaving behind the soluble impurities and insoluble particulates.
The main drawback of this technique is that it requires appreciable capital investments in design, construction and validation of high-gradient magnetic separators. Also, the technique does not lend itself to disposable applications, which are poised to become the norm for protein purification in the Bioprocess industry.
What is needed is a better process for purifying biomolecules in fewer steps with fewer materials.